The axon initial segment (AIS) is the site of action potential initiation in most vertebrate neurons, and is thus a critical control point for the regulation of neuronal excitability. Mutations in ion channels that alter AIS excitability cause nervous system diseases such as epilepsy, but we do not yet have a detailed understanding of key aspects of AIS structure and function. Most areas of basic neurobiology have benefited from the variety of approaches available in both vertebrate and invertebrate model systems. However, AIS research has not benefited from the advanced genetic tools, including forward genetic screening, available in Drosophila and other model invertebrates because of the prevailing view that the AIS is a unique feature of vertebrate neurons. The use of the Drosophila system has yielded numerous critical insights into diverse aspects of neurobiology including axon guidance, neuronal differentiation and development, synaptic function and ion channel signaling, and would almost certainly provide new insights into AIS function if it could be used. In this proposal, we aim to build on our recent findings that the proximal axons of Drosophila sensory neurons have striking and unexpected similarities on the molecular level to the vertebrate AIS. We aim to (1) demonstrate that Drosophila sensory neurons do, in fact, have a fully functional AIS, and to (2) generate tools to facilitate molecular genetic dissection of AIS function in a way that has not previously been possible. Our preliminary data shows that the proximal axon of Drosophila sensory neurons shares key features of the vertebrate AIS, including a giant ankyrin-based cytoskeleton that forms a diffusion barrier separating the axonal compartment and concentrates voltage-gated ion channels. In this proposal, we seek to build on these data and establish that Drosophila sensory neurons have an AIS fully orthologous to that of vertebrate neurons. We will focus on determining which voltage-gated ion channels localize to the proximal axon, how they are recruited to this location, and whether the proximal axon is indeed the site of action potential initiation. This work and the reagents generated to complete it (principally a set of fly lines expressing green fluorescent protein-tagged ion channels) when used in combination with state-of-the-art optical sensors of neuronal function, will enable future genome-wide screens of AIS structure and regulation in live animals. We will thus lay the groundwork for future studies of the role of each channel in controlling neuronal output, as well as enabling screens to identify factors that regulate the interplay between ion channels and the cytoskeleton at the AIS. This toolkit will be unparalleled in any other model system and will likely lead to important new insights into the role of the AIS in neuronal signaling.